The present invention relates to methods for capturing samples for evaluation. More particularly, the present invention relates to an approach which allows the collection and concentration of microbes, possessing genes encoding specific enzymes or small molecule pathways, from complex or dilute microbial populations in aqueous or terrestrial environments.
There is a critical need in the chemical industry for efficient catalysts for the practical synthesis of optically pure materials; enzymes can provide the optimal solution. All classes of molecules and compounds that are utilized in both established and emerging chemical, pharmaceutical, textile, food and feed, detergent markets must meet stringent economical and environmental standards. The synthesis of polymers, pharmaceuticals, natural products and agrochemicals is often hampered by expensive processes which produce harmful byproducts and which suffer from low enantioselectivity. Enzymes have a number of remarkable advantages which can overcome these problems in catalysis: they act on single functional groups, they distinguish between similar functional groups on a single molecule, and they distinguish between enantiomers. Moreover, they are biodegradable and function at very low mole fractions in reaction mixtures. Because of their chemo-, regio- and stereospecificity, enzymes present a unique opportunity to optimally achieve desired selective transformations. These are often extremely difficult to duplicate chemically, especially in single-step reactions. The elimination of the need for protection groups, selectivity, the ability to carry out multi-step transformations in a single reaction vessel, along with the concomitant reduction in environmental burden, has led to the increased demand for enzymes in chemical and pharmaceutical industries. Enzyme-based processes have been gradually replacing many conventional chemical-based methods. A current limitation to more widespread industrial use is primarily due to the relatively small number of commercially available enzymes. Only xcx9c300 enzymes (excluding DNA modifying enzymes) are at present commercially available from the  greater than 3000 non DNA-modifying enzyme activities thus far described.
The use of enzymes for technological applications also may require performance under demanding industrial conditions. This includes activities in environments or on substrates for which the currently known arsenal of enzymes was not evolutionarily selected. Enzymes have evolved by selective pressure to perform very specific biological functions within the milieu of a living organism, under conditions of mild temperature, pH and salt concentration. For the most part, the non-DNA modifying enzyme activities thus far described have been isolated from mesophilic organisms, which represent a very small fraction of the available phylogenetic diversity. The dynamic field of biocatalysis takes on a new dimension with the help of enzymes isolated from microorganisms that thrive in extreme environments. Such enzymes must function at temperatures above 100xc2x0 C. in terrestrial hot springs and deep sea thermal vents, at temperatures below 0xc2x0 C. in arctic waters, in the saturated salt environment of the Dead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 11 in sewage sludge. Enzymes obtained from these extremophilic organisms open a new field in biocatalysis.
For example, several esterases and lipases cloned and expressed from extremophilic organisms are remarkably robust, showing high activity throughout a wide range of temperatures and pHs. The fingerprints of five of these esterases show a diverse substrate spectrum, in addition to differences in the optimum reaction temperature. As seen in FIG. 1, esterase 5 (EST5) recognizes only short chain substrates while esterase 2 (EST2) only acts on long chain substrates in addition to a significant difference in the optimal reaction temperature. These results suggest that more diverse enzymes fulfilling the need for new biocatalysts can be found by screening biodiversity.
Furthermore, virtually all of the enzymes known so far have come from cultured organisms, mostly bacteria and more recently archaea. Traditional enzyme discovery programs rely solely on cultured microorganisms for their screening programs and are thus only accessing a small fraction of natural diversity. Several recent studies have estimated that only a small percentage, conservatively less than 1%, of organisms present in the natural environment have been cultured (see Table I). Amann et al., Microbiol. Rev. 59:143 (1995); Barnes et al., Proc. Natl. Acad. Sci. 91:1609 (1994); Torvisk et al., Appl. Environm. Microbiol. 56:782 (1990). Hence, this vast majority of microorganisms represents an untapped resource for the discovery of novel biocatalysts.
Within the last decade there has also been a dramatic increase in the need for bioactive compounds with novel activities. This demand has arisen largely from changes in worldwide demographics coupled with the clear and increasing trend in the number of pathogenic organisms that are resistant to currently available antibiotics. For example, while there has been a surge in demand for antibacterial drugs in emerging nations with young populations, countries with aging populations, such as the US, require a growing repertoire of drugs against cancer, diabetes, arthritis and other debilitating conditions. The death rate from infectious diseases has increased 58% between 1980 and 1992 and it has been estimated that the emergence of antibiotic resistant microbes has added in excess of $30 billion annually to the cost of health care in the US alone. As a response to this trend pharmaceutical companies have significantly increased their screening of microbial diversity for compounds with unique activities or specificities.
There are several common sources of lead compounds (drug candidates), including natural product collections, synthetic chemical collections, and synthetic combinatorial chemical libraries, such as nucleotides, peptides, or other polymeric molecules. Each of these sources has advantages and disadvantages. The success of programs to screen these candidates depends largely on the number of compounds entering the programs, and pharmaceutical companies have to date screened hundred of thousands of synthetic and natural compounds in search of lead compounds. Unfortunately, the ratio of novel to previously-discovered compounds has diminished with time. The discovery rate of novel lead compounds has not kept pace with demand despite the best efforts of pharmaceutical companies. There exists a strong need for accessing new sources of potential drug candidates.
The majority of bioactive compounds currently in use are derived from soil microorganisms. Many microbes inhabiting soils and other complex ecological communities produce a variety of compounds that increase their ability to survive and proliferate. These compounds are generally thought to be nonessential for growth of the organism and are synthesized with the aid of genes involved in intermediary metabolism hence their namexe2x80x94xe2x80x9csecondary metabolitesxe2x80x9d. Secondary metabolites that influence the growth or survival of other organisms are known as xe2x80x9cbioactivexe2x80x9d compounds and serve as key components of the chemical defense arsenal of both micro- and macroorganisms. Humans have exploited these compounds for use as antibiotics, antiinfectives and other bioactive compounds with activity against a broad range of prokaryotic and eukaryotic pathogens. Approximately 6,000 bioactive compounds of microbial origin have been characterized, with more than 60% produced by the gram positive soil bacteria of the genus Streptomyces. Of these, at least 70 are currently used for biomedical and agricultural applications. The largest class of bioactive compounds, the polyketides, include a broad range of antibiotics, immunosuppressants and anticancer agents which together account for sales of over $5 billion per year.
Despite the seemingly large number of available bioactive compounds, it is clear that one of the greatest challenges facing modern biomedical science is the proliferation of antibiotic resistant pathogens. Because of their short generation time and ability to readily exchange genetic information, pathogenic microbes have rapidly evolved and disseminated resistance mechanisms against virtually all classes of antibiotic compounds. For example, there are virulent strains of the human pathogens Staphylococcus and Streptococcus that can now be treated with but a single antibiotic, vancomycin, and resistance to this compound will require only the transfer of a single gene, vanA, from resistant Enterococcus species for this to occur. When this crucial need for novel antibacterial compounds is superimposed on the growing demand for enzyme inhibitors, immunosuppressants and anti-cancer agents it becomes readily apparent why pharmaceutical companies have stepped up their screening of microbial diversity for bioactive compounds with novel properties. There is still tremendous biodiversity that remains untapped as the source of lead compounds.
The present invention provides a path to access biodiversity for a variety of purposes, including the use in the eventual discovery of novel bioactivities.
The present invention provides a means for selectively attracting microbes to specific substrates chemically conjugated to a solid surface. The invention further provides for the enrichment of these microbes. This approach allows for the concentration and collection of microbes, possessing genes encoding specific enzymes or small molecule pathways, from complex or dilute microbial populations in aqueous or terrestrial environments. The basis for the attraction and subsequent enrichment is that microbes possess specific receptors that signal chemotactic attraction towards specific substrates. By binding the substrate to a surface and subsequently incubating the substrate-surface conjugate in the presence of a mixed microbial population, specific members of that population can be collected.
It is an object of the present invention to provide a means for selectively enriching for specific microorganisms from the surrounding environmental matrix. In accomplishing these and other objects, there has been provided, in accordance with one aspect of the present invention, a device for collecting a population of microorganisms from an environmental sample comprising a solid support having a surface for attaching a selectable microbial enrichment media.
In one aspect of the invention, microbial enrichment media containing a microbial attractant is used to selectively lure members of the environmental community to the device. In another aspect of the invention, bioactive compounds which inhibit the growth of unwanted organisms is included in the microbial enrichment media to further enhance selection of desirable microorganisms.
In yet another aspect of the invention, a method for isolating microorganisms from an environmental sample comprising contacting the sample with a device having a solid support and a surface for attaching a selectable microbial enrichment media and isolating the population from the device is provided.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.